Identification of the Magnesium Ion Binding Site in the Catalytic Center of Escherichia coli Primase by Iron Cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg2+ binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg2+ ions were replaced with Fe2+ ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe2+ cleavages were generated at sites designated I, II, and III. Adding Mg2+ or Mn2+ ions to the reaction strongly inhibited Fe2+ cleavage; however, adding Ca2+ or Ba2+ ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg2+ ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.